Generation of an Fsp1 (fibroblast‐specific protein 1)‐Flpo transgenic mouse strain

Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases (Flp, Flpe, and Flpo) bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1‐Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of Fsp1 (fibroblast‐specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1‐Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype.     

Gene transfer to skeletal muscle using hydrodynamic limb vein injection: current applications,hurdles and possible optimizations

Hydrodynamic limb vein injection is an in vivo locoregional gene delivery method. It consists of administrating a large volume of solution containing nucleic acid constructs in a limb with both blood inflow and outflow temporarily blocked using a tourniquet. The fast, high pressure delivery allows the musculature of the whole limb to be reached. The skeletal muscle is a tissue of choice for a variety of gene transfer applications, including gene therapy for Duchenne muscular dystrophy or other myopathies, as well as for the production of antibodies or other proteins with broad therapeutic effects. Hydrodynamic limb vein delivery has been evaluated with success in a large range of animal models. It has also proven to be safe and well‐tolerated in muscular dystrophy patients, thus supporting its translation to the clinic. However, some possible limitations may occur at different steps of the delivery process. Here, we have highlighted the interests, bottlenecks and potential improvements that could further optimize non‐viral gene transfer following hydrodynamic limb vein injection.     

The coordination of spindle‐positioning forces during the asymmetric division of the Caenorhabditis elegans zygote

In Caenorhabditis elegans zygote, astral microtubules generate forces essential to position the mitotic spindle, by pushing against and pulling from the cortex. Measuring microtubule dynamics there, we revealed the presence of two populations, corresponding to pulling and pushing events. It offers a unique opportunity to study, under physiological conditions, the variations of both spindle‐positioning forces along space and time. We propose a threefold control of pulling force, by polarity, spindle position and mitotic progression. We showed that the sole anteroposterior asymmetry in dynein on‐rate, encoding pulling force imbalance, is sufficient to cause posterior spindle displacement. The positional regulation, reflecting the number of microtubule contacts in the posterior‐most region, reinforces this imbalance only in late anaphase. Furthermore, we exhibited the first direct proof that dynein processivity increases along mitosis. It reflects the temporal control of pulling forces, which strengthens at anaphase onset following mitotic progression and independently from chromatid separation. In contrast, the pushing force remains constant and symmetric and contributes to maintaining the spindle at the cell centre during metaphase.     

Association of CAT polymorphisms with catalase activity and exposure to environmental oxidative stimuli

We tested the hypotheses that catalase activity is modified by CAT single nucleotide polymorphisms (SNPs) (-262;-844), and by their interactions with oxidant exposures (coal dusts, smoking), lymphotoxin alpha (LTA, NcoI) and tumor necrosis factor (TNF, -308) in 196 miners. Erythrocyte catalase, superoxide dismutase, and glutathione peroxidase activities were measured. The CAT -262 SNP was related to lower catalase activity (104, 87 and 72 k/g hemoglobin for CC, CT and TT, respectively, p < 0.0001). Regardless of CAT SNPs, the LTA NcoI but not the TNF-308 SNP was associated with catalase activity (p = 0.04 and p = 0.8). CAT -262 T carriers were less frequent in highly exposed miners (OR = 0.39 [0.20–0.78], p = 0.007). In CAT -262 T carriers only, catalase activity decreased with high dust exposure (p = 0.01). Haplotype analyses (combined CAT SNPs) confirm these results. Results show that CAT -262 and LTA NcoI SNPs, and interaction with coal dust exposure, influenced catalase activity.     

Cyclooxygenase-2 positively regulates Akt signalling and enhances survival of erythroleukemia cells exposed to anticancer agents

Cyclooxygenase-2 (COX-2) has been found to be highly expressed in many types of cancers and to contribute to tumorigenesis via the inhibition of apoptosis, increased angiogenesis and invasiveness. In hematological malignancies, COX-2 expression was found to correlate with poor patient prognosis. However, the exact role of COX-2 expression in these malignancies, and particularly in erythroleukemias, remains unclear. The aim of this work was to describe and understand the relationships between COX-2 expression and apoptosis rate in erythroleukemia cells after apoptosis induction by several anticancer agents. We used three different erythroleukemia cell lines in which COX-2 expression was modulated by transfection with either COX-2 siRNA or COX-2 cDNA. These cellular models were then treated with apoptosis inducers and apoptosis onset and intensity was followed. Cell signalling was evaluated in unstimulated transfected cells or after apoptosis induction. We found that COX-2 inhibition rendered erythroleukemia cells more sensitive to apoptosis induction and that in cells overexpressing COX-2 apoptosis induction was reduced. We demonstrated that COX-2 inhibition decreased the pro-survival Akt signalling and activated the negative regulator of Akt signalling, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Conversely, in COX-2 overexpressing cells, Akt signalling was activated and PTEN was inhibited. In these last cells, inhibition of casein kinase 2 or Akt signalling restored sensitivity to apoptotic agents. Our findings highlighted that COX-2 can positively regulate Akt signalling mostly through PTEN inhibition, partly via casein kinase 2 activation, and enhances survival of erythroleukemia cells exposed to anticancer agents.     

Subthalamic Nucleus High-Frequency Stimulation Restores Altered Electrophysiological Properties of Cortical Neurons in Parkinsonian Rat

Electrophysiological recordings performed in parkinsonian patients and animal models have confirmed the occurrence of alterations in firing rate and pattern of basal ganglia neurons, but the outcome of these changes in thalamo-cortical networks remains unclear. Using rats rendered parkinsonian, we investigated, at a cellular level in vivo, the electrophysiological changes induced in the pyramidal cells of the motor cortex by the dopaminergic transmission interruption and further characterized the impact of high-frequency electrical stimulation of the subthalamic nucleus, a procedure alleviating parkinsonian symptoms. We provided evidence that a lesion restricted to the substantia nigra pars compacta resulted in a marked increase in the mean firing rate and bursting pattern of pyramidal neurons of the motor cortex. These alterations were underlain by changes of the electrical membranes properties of pyramidal cells including depolarized resting membrane potential and increased input resistance. The modifications induced by the dopaminergic loss were more pronounced in cortico-striatal than in cortico-subthalamic neurons. Furthermore, subthalamic nucleus high-frequency stimulation applied at parameters alleviating parkinsonian signs regularized the firing pattern of pyramidal cells and restored their electrical membrane properties.     

Characterization of a surrogate murine antibody to model anti-human CD3 therapies

Fc-modified anti-human CD3ε monoclonal antibodies (mAbs) are in clinical development for the treatment of autoimmune diseases. These next generation mAbs have completed clinical trials in patients with type-1 diabetes and inflammatory bowel disease demonstrating a narrow therapeutic window. Lowered doses are ineffective, yet higher pharmacologically-active doses cause an undesirable level of adverse events. Thus, there is a critical need for a return to bench research to explore ways of improving clinical outcomes. Indeed, we recently reported that a short course of treatment affords synergy, providing long-term disease amelioration when combining anti-mouse CD3 and anti-mouse tumor necrosis factor mAbs in experimental arthritis. Such strategies may widen the window between risk and benefit; however, to more accurately assess experimentally the biology and pharmacology, reagents that mimic the current development candidates were required. Consequently, we engineered an Fc-modified anti-mouse CD3ε mAb, 2C11-Novi. Here, we report the functional characterization of 2C11-Novi demonstrating that it does not bind FcγR in vitro and elicits little cytokine release in vivo, while maintaining classical pharmacodynamic effects (CD3-TCR downregulation and T cell killing). Furthermore, we observed that oral administration of 2C11-Novi ameliorated progression of remitting-relapsing experimental autoimmune encephalitis in mice, significantly reducing the primary acute and subsequent relapse phase of the disease. With innovative approaches validated in two experimental models of human disease, 2C11-Novi represents a meaningful tool to conduct further mechanistic studies aiming at exploiting the immunoregulatory properties of Fc-modified anti-CD3 therapies via combination therapy using parenteral or oral routes of administration.     

The Effect of Three-Monthly Albendazole Treatment on Malarial Parasitemia and Allergy: A Household-Based Cluster-Randomized,Double-Blind,Placebo-Controlled Trial

Background

Helminth infections are proposed to have immunomodulatory activities affecting health outcomes either detrimentally or beneficially. We evaluated the effects of albendazole treatment, every three months for 21 months, on STH, malarial parasitemia and allergy.

Methods and Findings

A household-based cluster-randomized, double-blind, placebo-controlled trial was conducted in an area in Indonesia endemic for STH. Using computer-aided block randomization, 481 households (2022 subjects) and 473 households (1982 subjects) were assigned to receive placebo and albendazole, respectively, every three months. The treatment code was concealed from trial investigators and participants. Malarial parasitemia and malaria-like symptoms were assessed in participants older than four years of age while skin prick test (SPT) to allergens as well as reported symptoms of allergy in children aged 5–15 years. The general impact of treatment on STH prevalence and body mass index (BMI) was evaluated. Primary outcomes were prevalence of malarial parasitemia and SPT to any allergen. Analysis was by intention to treat. At 9 and 21 months post-treatment 80.8% and 80.1% of the study subjects were retained, respectively. The intensive treatment regiment resulted in a reduction in the prevalence of STH by 48% in albendazole and 9% in placebo group. Albendazole treatment led to a transient increase in malarial parasitemia at 6 months post treatment (OR 4.16(1.35–12.80)) and no statistically significant increase in SPT reactivity (OR 1.18(0.74–1.86) at 9 months or 1.37 (0.93–2.01) 21 months). No effect of anthelminthic treatment was found on BMI, reported malaria-like- and allergy symptoms. No adverse effects were reported.

Conclusions

The study indicates that intensive community treatment of 3 monthly albendazole administration for 21 months over two years leads to a reduction in STH. This degree of reduction appears safe without any increased risk of malaria or allergies.

Trial Registration

Controlled-Trials.com ISRCTN83830814     

Male Gender,Increased Blood Viscosity,Body Mass Index and Triglyceride Levels Are Independently Associated with Systemic Relative Hypertension in Sickle Cell Anemia

Patients with sickle cell anemia (SCA) have usually lower diastolic, systolic and mean blood pressure (BP) than the general population. However, BP values ≥120/70 mmHg considerably increase the risk for acute and chronic complications in SCA. The aim of this study was to identify biological factors associated with relative hypertension in adults with SCA. We compared the hematological, lipid and hemolytic profiles, as well as blood viscosity, between SCA patients with normal BP (<120/70 mmHg, n = 54) and those with relative hypertension (BP≥120/70 mmHg, n = 43). Our results demonstrated that male gender (OR: 3.49; 95%CI 1.20 to 10.16, p<0.05), triglycerides (OR: 9.19; 95% CI 2.29 to 36.95, p<0.01), blood viscosity (OR: 1.35; 95% CI 1.01 to 1.81, p<0.05) and body mass index (OR: 1.37; 95% CI 1.14 to 1.64, p<0.01) were independent risks factors for relative hypertension in SCA. No association was found between the BP status and the positive history of painful vaso-occlusive crisis or acute chest syndrome. An association between triglycerides level and the occurrence of these two major acute complications was detected. Our study suggests that male gender, increased triglycerides level, BMI and blood viscosity could increase the risk for developing relative hypertension in SCA. In addition, our results support a role of moderately elevated triglycerides in the pathophysiology of vaso-occlusive events.